2nd Conference of Scientific Association of Animal Health Research Institute, 2-6/2/2014
pp. 142-157 ISSN: 2356-7767
Evaluation of the antimicrobial effect of zinc oxide nanoparticles on Listeria monocytogenes and Candida albicans isolated from infected Egyptian buffalo suffering from abortion Nahed, M. A. Shawky *; Atef, A. Hassan**; Rasha, M.H. Sayed El Ahl** and H.H. Mahmoud*** *Buffalo Diseases Research Department; **Mycology and Mycotoxins Department, Animal Health Research Institute, Dokki, Cairo, Egypt and ***Central Laboratory Of Elemental and Isotopic Analysis, Nuclear Research Centre, Atomic Energy Authority, Egypt.
[email protected] Received in 12/1/2014 Accepted in 19/1/2014 Abstract Out o..f 40 cases of buffalo’s abortion in private farm at Assuit governorate, 160 samples were collected included milk, vaginal swabs, placenta and fetal stomach content (40 of each). L. monocytogenes was recovered from most of collected samples at relatively higher rates than the isolated C.albicans; where they detected in 45.5% and 20 % of milk samples, 20 and 7.5%of placenta, 30 and 20% of fetal stomach contents, respectively. Whereas, C.albicans was recovered in a comparatively higher number of samples of vaginal swabs (from 50%) than L.monocytogenes (from 40%). Biosynthesis of zinc oxide nanoparticles (ZnO-NPs) were conducted using C.albicans ,characterized for their optical and Nano structural properties using UV-Vis Spectrophotometer. The external morphology and particle size were characterized by Scanning Electron Microscope (SEM). The prepared ZnO-NPS was evaluated for its antimicrobial potentials against L.monocytogenes and C. albicans using the technique of well and disc diffusion test. The minimum inhibitory concentration (MIC) of ZnO-NPS for C.albicans was100 µg/ml in case of well diffusion test but it was 50µg/ml in disc diffusion tests. Whereas, for L.monocytogenes the MIC required higher concentration of nanoparticles to obtain satisfactory inhibition for their growth (3.0 mg/ml or more) compared with known antibacterial and antifungal. The changes in cells membrane of treated L.monocytogenes and C. albicans with ZnO-NPs were evaluated and observed by scanning electron microscope (SEM). Therefore, ZnO-NPs could be used in drug delivery for successful treatment of microbial diseases of human and animal. The biosynthesis of metal nanoparticles was highly biocompatible, cheap and environmental friendly. Key words: L.monocytogenes, C.albicans, abortion, antimicrobial effect of ZnO-NPs. sumption. One of essential animals in our country included buffalo (Bubalus bubalis) which considered as “black gold”. The products of these animals of huge economical importance in developing countries as Egypt (milk, meat, hides and draft power for various agricultural operations (Patel et al., 2010). Hence, infectious abortion in buffalo which caused heavy economic losses; was of public
Introduction The new challenge of this century is the application of nanotechnology in new fields like food industry. Up to now nanotechnology focused on the medicine, drugs formulation, electronics, textiles, defense industry, cosmetics and agriculture (Reddy, et al., 2007). The animal wealth in developing country represents the major role in food security for human con142
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cells (Reddy et al., 2007). ZnO is 1 of 5 zinc compounds that are currently listed as Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration. Zinc salt has been used for the treatment of zinc deficiency (Lopes de Romana et al., 2002). ZnO nanoparticles have been shown to have a wide range of antibacterial activities against both Gram-positive and Gram-negative bacteria, including major foodborne pathogens like E. coli O157:H7, Salmonella, L.monocytogenes and S. aureus (Jones et al., 2008 and Liu et al., 2009). Therefore, the present study was undertaken to evaluate the antimicrobial effects of ZnO-NPs against the recovered L. monocytogenes and C. albicans from cases of abortion in Egyptian buffalo. The used ZnO-NPs were synthesized biologically by fungus itself as cheapest and environment friendly method to avoid the environmental pollution.
health concern and could be transmitted to human workers that contact to infected animals; where, the zoonotic infection rate was (63.3%) brucellosis in the butchers; and a combined infection rate of (31.8%) in humans were also noted (Cadmus et al., 2006). Various studies have been carried out to identify the causes of abortion in cow, which suggest that most frequently isolated agents, Brucellas pecies, Campylobacter spp., Leptospira spp., Listeria spp., Salmonella spp., Chlamydophila spp., Mycoplasma spp. and a variety of viruses and mycotic agents (Rocourt and Cossart, 1997 and Mccausl et al., 2008). L. monocytogenes can cause mastitis, metritis, keratoconjunctivitis, encephalitis' circling disease', reproductive diseases, and iritis (Lowand Donachie, 1997 and Sanchez et al., 2001). Previous study showed that the L. monocytogenes is a causative agent for 3.58% of the cases of bovine abortion (Kirkbride, 1993) which occur approximately one week after exposure, and occur most commonly during the last trimester of pregnancy, although they may occur as early as the 4th month of gestation, the aborted fetus is often autolyzed. The buffaloes may show clinical signs of disease as well, although once returned to healthy appear to resist re-infection (Anderson et al., 1990). On the other hand, Candidosis caused by several species of the genus Candida is the most wide spread and prevalent mycotic disease of man and animals (Odds and Bernaerts, 1994; Hassan et al., 2010). C. albicans had been incriminated as a cause of bovine mastitis (Stuart, 1951), bovine abortion (Austwicket al., 1966), systemic candidosis as well as pharyngitis and eosophogitis in calves (Mills and Hirth, 1967), endometritis in mares (Tatezawa, 1958) and mastitis in sheep and goats (Rezk, 1981 and Hassan et al., 2011). The mycotic abortion usually occur during the winter and spring months, since when animals are often kept in total confinement and can be exposed to moldy hay or silage. Recent studies have shown that these nanoparticles have selective toxicity to bacteria and fungi but exhibit minimal effects on human
Materials and Methods Sampling. A total of 160 samples of milk, vaginal swab, placenta and fetal stomach content (40 of each) were collected from 40 cases of buffalo abortion in a private farm at Assuit governorate, in sterile containers and swabs and transferred rapidly in ice box to the laboratory for investigation the incidence of L. monocytogenes and C. albican in abortion according to (Quinn et al., 2002). Antibacterial and antimycotic agents antibiotics. A known antibacterial and antifungal as Cefotaxime (30 µg/disc) and Fluconazole (10 µg/ml) were purchased from Al Gomhouria pharmaceutical company and used as a positive control toward bacteria and fungi, respectively. Isolation and identification of L. monocytogenes according to Sneath et al., (1989). A loopfull from the last prepared samples were streaked onto Palcam and Oxford media and incubated at 35°C for 24-48 h. Suspected colony were cultivated onto trypticase soy agar with yeast extract (TSA-YE) and incubated at 30°C for 24h, then examined microscopically for non-sporulated and capsulated Gram positive short rods. 143
2nd Conference of Scientific Association of Animal Health Research Institute, 2-6/2/2014
Biochemical and serological identification of L.monocytogenes (Sneath et al., 1989). Some tests were used to confirm the identification of L. monocytogenesasmotility test; biochemical test, blood hemolysis and Christie Atkins Munch-Petersen (CAMP) test (Anonymous, 2004). In addition, serological identification was carried out according to Federal Register, (1988) by agglutination test. Pathogenicity and virulence test of L.monocytogenes according to Federal Register, (1988) and Gutekunst et al., (1992). a- Using mice. 24 mice were used. They were divided into 8 groups (3 to each), each mice was injected intra-peritoneally (I/P) with 0.1 ml of L.monocytogenes supernatant protein starting by 103 till reach 109 CFU/ ml using Macferland’s tube. Three mice were kept as control and the number of deaths was recorded daily for each of 7 days. b- Using rabbit (Anton’s eye test). Each rabbit was infected with 0.1ml of the bacterial suspension (109) by instillation into the conjunctiva of one eye. Conjunctivitis within 24 hours was recorded. Isolation and identification of yeasts from samples. Samples were prepared and examined for isolation of yeast according to the technique recommended by (Refai, 1998 and Refai et al., 2012). All samples were processed and cultivated on different media including Sabouraud’s dextrose agar (SDA), brain heart infusion agar (BHIA), rice agar, at 37 °C for 2 - 5 days. The pure yeast isolates identified morphological using the slide mount technique, growth on rice agar medium, germ tube test. In addition, the yeast isolates were examined by sugar fermentation, sugar assimilation, nitrate reduction and urease tests. Biosynthesis of ZnO- Nanoparticles. A-Preparation of Mycelium and Supernatants of fungi (Hartsel and Bolard, 1996). The spore suspension of C. albicans(105/ml) was inoculated into 250 ml Erlenmeyer flasks, each containing 50mL of semi defined medium (SDM) composed of KH2PO4 (7g/L), K2HPO4 (2 g/L), MgSO4.7H2O (0.1 g/L), (NH4) 2SO4 (0.1 g/L), yeast extract (0.6 g/L), and glucose
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(10 g/L) at 30°C under shaking condition (200 rpm) for 96 hrs. Mycelia were separated from the culture broth by centrifugation at 4500 rpm, 10 °C, for 15 min. The settled mycelia were washed thrice with de-ionized water. B. Biosynthesis of ZnO Nanoparticles (Jha et al., 2009). The 1% of washed mycelia of C.albican was inoculated into tubes containing analytical reagent grade Zinc Chloride (ZnCl2) which was used for preparing a solution of 0.25(M) strength at room temperature. Control solution was prepared by adding 100 ml sterile distilled water, carbon and nitrogen containing nutrients and the mild base in known quantitative ratio (5:1:1). To each of these tubes, 20 ml of ZnCl2 solution was added. The pH of the control tube was noted to be 8-9. Culture solution containing tubes including control tube were heated on the steam bath up to 80°C for 5 to 10 minutes. An appearance of starch like haziness in solution and white deposition at the bottom of the tube was perceived as an indication of commencement of transformation. No such deposition or haziness was observed in control tube. The tubes were allowed to incubate in the laboratory ambience for another 9h, after which distinctly makeable coalescent white clusters deposited at the bottom of all the tubes except in control. A remarkable change in pH was observed at this stage (6.0 to 7.5) excluding control (8 to 9). C. Characterization of prepared ZnO-NPs
(Roisnel and Rodrıguez, 2000 and Awodugba and Ilyas, 2013). The prepared
ZnO-NPs were characterized for their optical and Nano structural properties. The optical absorption spectra of ZnO dispersed in water were recorded using UV-Vis Spectrophotometer (GENESYS 10s VL.200 2L7H311008). The external morphology and particle size of the preparation were characterized by using Transmission electron micrograph (TEM) HITACHI H-800 (Hitachi) and Scanning electron microscope (SEM) (Joe, JSM-5600LV, and Japan), FT-IR spectra (Fourier Transform Infrared Spectrometer) were recorded on Jasco FT-IR5300 model spectrophotometer in ..KBr pellets 144
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for 30 min). The agar blocks were dried with a critical-point drier using liquid CO2 and coated with gold-coater for 5 min. The coated samples were observed under JSM-5600LVwith accelerating voltage of 10 kV.
Antibacterial and antifungal activity of prepared ZnO-NPs (Jin et al., 2009). The prepared ZnO-NPs was evaluated for its antimicrobial activity against the isolates of L.monocytogenes and C. albicans that recovered from the present study. The minimum inhi bi t or y conc ent ra t i on (M IC ) for L.monocytogenes and C.albicans was determined by a disc and well diffusion methods. The media of BHIA and SDA were poured into plates containing one ml cell suspension of L. monocytogenes (5 х 104 cells /ml) and spores suspension of C. albicans, (2.5 х 104 cells/ml) respectively, shaken over the tables in rotary manner. After solidification, the medium plates either pored or its surface impregnated with discs for different concentration of Zno-NPs (50, 100& 150 µg/ml for C. albicans and 0.5, 3.0 and 5.0 mg/ml for L. monocytogenes) which also were added to separate pores of medium plates and it was incubated at 37◦C for 28 hrs. To establish the antimicrobial activity of ZnO-NPs on the bacterial and fungal growth, the MIC of ZnO-NPs was determined by optical density of the bacterial or fungal culture solution containing different concentration of each ZnO-NP after 24 h, 48, and 96 h. The inhibitory concentration was defined as the lowest concentration that inhibited the growth as determined by a comparison with the growth in the control wells or discs of Cefotaxime (30 µg/disc) and fluconazole (10 µg/ml) as a positive control toward bacteria and fungi respectively. Scanning Electron Microscopy (SEM), (Gong et al., 2006). The morphological changes of treated L. monocytogenes and C. albicansby ZnO-NPs were observed with a scanning electron microscope (SEM). Strains were prepared by cutting the agar, fixed for a minimum of 3h in 2.5% glutaraldehyde (100mM phosphate buffer solution, pH 7.2), and then fixed in1% osmium tetra oxide for 1h. The agar blocks were dehydrated through a graded series of ethanol (30, 50, 60, 70, 80, 90, 95, and 100%; each level was applied twice for 15 min each time) and ethanol: isoamyl acetate (3:1, 1:1, 1:3, and 100% isoamyl acetate twice
Results and Discussion Microorganisms, such as bacteria, moulds, yeasts and viruses, in the living environment are often pathogenic and cause severe infections in both human beings and animals (Reddy, et al., 2007). The infectious disease caused by the genus Listeria is known as listeriosis. This genus comprises six characterized species: L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi (Rocourt and Cossart, 1997); two of these species, L. monocytogenes and L. ivanovii, are potentially pathogenic. L.monocytogenes was first reported as a cause of bovine abortion in 1939 (Graham et al., 1939). This organism occurs commonly in the environment, and it has been isolated from the feces of normal humans and other animals (Weis and Seelger, 1975). It apparently multiplies in poorly preserved silage, which becomes a frequent although not the only source of infection. Infected buffalo often have fever and anorexia prior to aborting, and post parturient complications sometimes include retained placenta, pyometra, septicemia, and occasional death (Erdogan et al., 2001). Signs of central nervous system involvement seldom occur in a herd along with abortions. Listeria abortions occur at all stages of gestation, but fetuses
